芝麻素酚重组酶法糖苷化及其促进益生菌黏附定植的分子机制

Sesaminol glucoside has the potential prebiotic activity. However, the effect and mechanism of sesaminol glucoside on probiotic adhesion and colonization have not yet been elucidated. In our previous studies, sesaminol has been obtained from sesame cake, and we have also preliminarily proved that sesaminol has the promoting effect on proliferation and adhesion of probiotics in vitro. In the present study, recombinant dextransucrase and enzymatic technology were used to catalyze glucosidylation of sesaminol, and thus, different kinds of glucosidylation level of sesaminol glucoside with high purity and yield would be prepared. The promoting effect on the proliferation of enteric probiotics in vitro would be investigated by anaerobic fermentation culture system . The promoting effect of sesaminol glucoside on probiotic adhesion and colonization would also be explored by analyzing the adhesive capacity of probiotic and colonic epithelial cells. CRISPR-Cas9 gene editing technology would be exploited to explore the mechanisms of the promoting effect of sesaminol glucoside on the adhesion and colonization of probiotics, based on the adhesion molecule β-catenin on the surface of colonic epithelial cells. Furthermore, the effects on the domain of β-catenin related to probiotic adhesion would also be investigated. This study would provide new research ideas and methods for preparation of food-derived lignans by recombinant enzymatic glycosylation method, and for the molecular mechanism of promoting effect of prebiotics on adhesion and colonization of probiotics investigated by gene editing technology.

芝麻素酚葡萄糖苷具有潜在益生元活性，对其促进肠道益生菌增殖和黏附作用尚未阐明。我们前期研究已从芝麻粕中得到芝麻素酚单体，发现芝麻素酚在体外具有促进肠道益生菌增殖和黏附的作用。本项目拟采用酶法糖基化修饰技术，利用重组右旋糖酐蔗糖酶，催化芝麻素酚的糖苷化反应，以制备出高纯度、高收率及不同糖基化程度的芝麻素酚葡萄糖苷。采用体外厌氧发酵培养体系，研究其对肠道益生菌的增殖作用；通过分析益生菌与结肠上皮细胞的黏附能力，研究其促进肠道益生菌黏附的作用。进一步采用CRISPR-Cas9基因编辑技术，基于结肠上皮细胞表面黏附因子β-链蛋白(β-catenin)，研究芝麻素酚葡萄糖苷促进益生菌黏附定植的机制；并深入研究与益生菌黏附相关的β-catenin蛋白结构域。本研究将为食源性木脂素的重组酶法制备、以及利用基因编辑技术探索益生元促进益生菌黏附定植机制的研究，开创了新的研究思路和研究方法。

本项目采用酶法糖基化修饰技术，研究了基因工程重组右旋糖酐蔗糖酶表达和纯化、酶学性质和酶活力；利用重组右旋糖酐蔗糖酶催化芝麻素酚的糖苷化反应，制备并纯化出芝麻素酚葡萄糖苷，并对其进行结构鉴定研究。结果表明，构建了重组表达质粒、右旋糖酐蔗糖酶的基因工程菌株；通过酶催化反应得到产物芝麻素酚葡萄糖苷，通过结构鉴定表明得到的终产物主要为芝麻素酚三葡萄糖苷。.采用体外厌氧发酵培养体系和体内动物实验、高通量测序分析，研究了芝麻素酚葡萄糖苷化产物对肠道益生菌和肠道菌群结构的调节作用；通过体外细胞学和微生物学实验分析了益生菌与结肠上皮细胞的黏附性能，研究了芝麻素酚葡萄糖苷促进肠道益生菌黏附定植的作用及其机制。结果表明，芝麻素、芝麻素酚、芝麻素酚葡萄糖苷均具有促进肠道益生菌增殖和黏附、调节肠道菌群结构、改善肠道微生态环境、促进肠道健康的作用；其中促进益生菌黏附的作用机制与提高结肠上皮细胞黏附蛋白因子（β-catenin、E-cadherin、Occludin、Claudin-1）的表达密切相关；并进一步阐释了芝麻素酚、芝麻素酚葡萄糖苷调节肠道菌群结构和肠道益生活性的构效关系，即糖苷化可对食品功能因子的结构具有保护作用、继而提高其肠道益生活性的作用机制。.采用CRISPR-Cas9基因编辑技术，基于结肠上皮细胞表面黏附因子β-链蛋白(β-catenin)及其蛋白结构域，研究了芝麻素酚葡萄糖苷促进益生菌黏附定植的分子机制。构建了β-catenin基因敲除正常结肠上皮细胞模型，分析测定了益生菌与结肠上皮细胞黏附性；并以Flag为报告基因，构建了重组质粒转染β-catenin基因敲除结肠上皮细胞，分别表达β-catenin蛋白的N端、Arm段、C端氨基酸片段；进一步研究了与益生菌黏附相关的结肠上皮细胞β-catenin蛋白结构域。结果表明，芝麻素酚糖苷促进益生菌对结肠上皮细胞的黏附作用机制，与结肠上皮细胞β-catenin蛋白的表达、尤其是与β-catenin蛋白的Arm段氨基酸片段的表达密切相关。 .本研究为食源性木脂素的重组酶法制备、及利用基因编辑技术探索食品功能因子促进益生菌黏附定植机制的研究，提供了研究思路和方法。